Recently, conversion of biomass through saccharification and fermentation into ethanol or other useful products as a replacement for fossil fuels has garnered considerable attention. Because biomass is a renewable resource typically rich in polymers of hexoses and pentoses, it is a promising substrate for fermentation.
Biomass for such conversion processes may be potentially obtained from numerous different sources, including, for example: wood, paper, agricultural residues, food waste, herbaceous crops, and municipal and industrial solid wastes to name a few.
Biomass is made up primarily of cellulose and hemicellulose bound up with lignin. The lignin inhibits the conversion of the biomass into ethanol or other biofuels, and, as a result, typically a pretreatment step is required to expose the polysaccharides, cellulose and hemicellulose. Once hemicellulose and cellulose are exposed, saccharification, either enzymatic or chemical, may be performed to break the polysaccharides into their constituent monosaccharide monomers. Pretreatment and saccharification are used, therefore, to break down the long polysaccharide chains and free the sugars before they are fermented for biofuel production. Fermentation can begin once free sugars are present, either because they are naturally present or because a portion of the biomass has been reduced to its component sugars, or both.
In order to be effective, current pretreatment and saccharification processes attempt to liberate the biomass sugars while also minimizing the formation of secondary products from the degradation of hemicellulose, cellulose, and lignin, because of the inhibitory effects secondary products may have on the subsequent fermentation processes. The presence of inhibitory secondary products has historically complicated ethanol production and increased the cost of production due to elaborate detoxification steps.
Although numerous techniques for pretreatment and saccharification exist, the most popular methods, and the most cost effective methods, including acid hydrolysis, produce secondary products in addition to sugars, that are inhibitory to fermentation. Inhibitory secondary products created as a result of the degradation of hemicellulose pentoses and hexoses include furfural and 5-hydroxymethylfurfural (HMF), respectively. Furfural and HMF may further be broken down into levulinic, acetic, and formic acids. Other inhibitory secondary products include phenolic compounds produced from the degradation of lignin and acetic acid produced by cleavage of acetyl groups within the hemicellulose. Concentrations of inhibitory secondary products in the hydrolysate will vary based on the source of the biomass and the hydrolysis method used.
Some of the secondary products formed from the breakdown of hemicellulose, cellulose and lignin are in themselves valuable substances. The inventors have realized that recovery of high-value secondary products from the hydrolysate can improve the economics of the biomass to biofuel process.
Other secondary products are not formed from chemical decomposition, but may be extracted from the biomass during pretreatment and hydrolysis. These extracted secondary products include terpenes, sterols, fatty acids, and resin acids. These extracted compounds may also be inhibitory to fermentation.
Inhibitory secondary products may be detrimental to the fermentation process, particularly as their concentration increases. Thus, it would be advantageous if a process could be developed that allows specific microbes, like yeast for example, to efficiently convert biomass hydrolysate into biofuels, such as ethanol, in the presence of inhibitory secondary products formed during pretreatment and hydrolysis.
Many inhibitory products have compound impacts when present with other inhibitory compounds; thus, a non-inhibitory amount of a certain compound may become inhibitory in the presence of a second inhibitory compound. Furthermore, even following partial recovery and/or removal of inhibitory secondary products, the remaining concentrations may be inhibitory to fermentation due to these synergies. Thus, it would be advantageous if a process could be developed that allows specific microbes, like yeast for example, to efficiently convert biomass hydrolysate into biofuels, such as ethanol, in the presence of inhibitory secondary products formed during pretreatment and hydrolysis, even when the concentrations of the individual inhibitory secondary products are below their respective inhibitory concentration level but their combined concentration is inhibitory.
Cellulose is a homogeneous polysaccharide composed of linearly linked glucose units. Glucose is a hexose, which may be readily fermented by a number of microbes including Saccharomyces cerevisiae (traditional baker's yeast) and Kluyveromyces marxianus. Yeast cells are especially attractive for cellulosic ethanol processes, as they have been used in biotechnology for hundreds of years, are tolerant to high ethanol and inhibitor concentrations, and can grow at low pH values. A low pH value helps avoid bacterial contamination and is therefore advantageous.
Unlike cellulose, hemicellulose is a heterogeneous polymer of pentoses, hexoses, and uronic acids. The saccharides principally found in hemicellulose are the pentoses xylose and arabinose and the hexoses glucose, mannose and galactose. The relative amounts of different pentoses and hexoses vary with the biomass type. The hemicellulose content of some cellulosic biomass may reach as high as 38% or more of the total dry biomass weight. Therefore, hemicelluloses, and the pentoses and hexoses they contain, may comprise a substantial portion of the convertible sugars available in the biomass. As a result, in order to improve the economics of the biomass to biofuel conversion process, much research has been performed on identifying microorganisms that efficiently convert pentoses and hexoses to biofuel, such as ethanol.
While numerous microbes have been found to process hexoses into ethanol, efficiently fermenting pentoses has proven more elusive. Some bacteria and fungi can inefficiently convert pentoses to ethanol and many microbes can only process pentoses when assisted by enzymes. For a long time it was thought that yeast strains could not anaerobically ferment pentoses. However, U.S. Pat. No. 4,359,534 to Kurtzman et al. discloses the use of Pachysolen tannophilus to ferment pentoses. Similarly, U.S. Pat. No. 7,344,876 to Levine discloses a pure culture of Kluyveromyces marxianus capable of proliferation on pentoses as the sole carbon source.
While the patents to Kurtzman and Levine disclose the use of yeasts for fermentation of pentoses into ethanol, commercial applications have been limited because of the detrimental effects of inhibitory secondary products typically found in biomass hydrolysate. Yeasts that can ferment xylose and other pentoses in an artificial, or controlled, medium generally perform poorly in acid hydrolysates. Challenges presented by biomass hydrolysate include an acidic pH and a high concentration of toxic compounds, including acetic acid, phenolic compounds, 5-hydroxymethylfurfural (HMF) and furfural, and other inhibitory molecules produced during hemicellulose hydrolysis.
Because of the detrimental effects of inhibitory secondary products on the production of ethanol, biomass hydrolysate is currently subjected to a conditioning process after pretreatment and hydrolysis to reduce the concentration of inhibitory secondary products. This conditioning process adds complexity and cost to the overall process and reduces the efficiency and cost-effectiveness of the conversion process. Furthermore, the greater the required reduction in the concentration levels of the inhibitory secondary products, the greater the complexity and cost. A need, therefore, exists for a process in which microbes, such as different yeast strains, could more effectively convert pentoses, as well as hexoses, into ethanol and other biofuels in the presence of inhibitors formed during the pretreatment and hydrolysis process. In addition, it would be beneficial to develop schemes whereby inhibitory secondary products may be partially recovered and purified, instead of only removed and discarded, from hydrolysate.
Furthermore, if an efficient method for converting pentoses to ethanol existed, the discarded hemicellulose in the paper pulping process might be converted into alcohol instead. Similarly, sugar cane residues, referred to as bagasse, could also be subjected to hemicellulose conversion prior to being combusted for their fuel values. The possibility of removing hemicellulose from the paper pulping process and converting it to ethanol was hypothesized by the Georgia Institute of Technology in W. J. Fredrick et al., Co production of ethanol and cellulose fiber from Southern Pine: A technical and economic assessment, 32 Biomass and Bioenergy 1293-1302 (2008). However, the Georgia Institute of Technology process explicitly requires the hydrolysate to be conditioned to remove inhibitors and noted the lack of an efficient process to convert pentoses into ethanol. The study noted that “Fermentation is carried out after inhibiting contaminants have been removed from the hydrolysate.” The study further notes that the 85% conversion factor of pentoses to ethanol “is an optimistic estimate that assumes that on-going research will make it possible . . . ” The study concludes that ethanol production from loblolly pine may not be competitive with ethanol from other lignocellulosic sources when it is co-produced with cellulose fiber.